Enterococcus faecalis is a gram-positive, facultatively anaerobic, coccal bacterium that causes a variety of community- and hospital- acquired infections in humans, including infections of the blood, endocardium, genitourinary tract, abdomen, wounds, and skin and soft tissue (burns, decubitis ulcers, diabetic foot ulcers). Preliminary studies in this laboratory have demonstrated that at least two E. faecalis isolates are able to survive in mouse peritoneal macrophages, J774A.1 macrophage-like cells, and murine bone marrow derived macrophages for an extended period of time. The long term goal of these studies is to obtain a better understanding of the interaction of E. faecalis with macrophages. The specific aims of this proposal are as follows: 1) to compare the survival of E. faecalis blood isolates, fecal isolates, and isogenic mutants lacking either cytolysin, gelatinase (protease) aggregation substance, UV resistance, or surface carbohydrate for the ability to survive in resident and elicited mouse peritoneal macrophages, 2) to compare the survival of E. faecalis when infected in vivo in peritoneal macrophages of BALB/c (Ity/s) and congenic BALB/c.DBA/2 (Ity/r) mice, 3) to determine the localization of E. faecalis in organs of infected mice and to determine the effect of neutrophil and macrophage depletion on the outcome of E. faecalis and persistence in the liver and spleen, 4) to generate a library of E. faecalis genomic insertional mutants and to screen these mutants for the loss of ability to survive in macrophages, and 5) to initiate characterizations of the insertionally-inactivated-E. faecalis genes that encode macrophage survival factors.